Apostolos P. Apostolidis

Mitochondrial DNA sequence variation and phylogeography among Salmo trutta L. (Greek brown trout) populations

To investigate the phylogenetic relationships and geographical structure among brown trout S. trutta L. populations from the South Adriatic‐Ionian and Aegean sea basins, mitochondrial DNA nucleotide sequence comparisons were used. A 310‐base‐pair (bp) segment of the control region (D‐loop), and an additional 280‐bp segment of the cytochrome b gene were sequenced from representatives of 13 brown trout populations. Phylogenetic analyses, conducted after combining the data presented with published data from other Eurasian brown trout, revealed four major phylogenetic groups, three of which were found widely distributed within the southern Balkan region. The phylogeographical patterns revealed by mtDNA represent one of the few cases where phylogenetic discontinuity in a gene tree exists without obvious geographical localization within a species range and has most likely resulted from the differentiation of the major mtDNA clades during Messinian or early Pleistocene times. Finally, the genetic relationships among the populations suggested by mtDNA were generally not in accordance with either allozyme or morphological data.

Quantitative Trait Loci Involved in Sex Determination and Body Growth in the Gilthead Sea Bream (Sparus aurata L.) through Targeted Genome Scan

Among vertebrates, teleost fish exhibit a considerably wide range of sex determination patterns that may be influenced by extrinsic parameters. However even for model fish species like the zebrafish Danio rerio the precise mechanisms involved in primary sex determination have not been studied extensively. The zebrafish, a gonochoristic species, is lacking discernible sex chromosomes and the sex of juvenile fish is difficult to determine. Sequential protandrous hermaphrodite species provide distinct determination of the gender and allow studying the sex determination process by looking at the mechanism of sex reversal. This is the first attempt to understand the genetic basis of phenotypic variation for sex determination and body weight in a sequential protandrous hermaphrodite species, the gilthead sea bream (Sparus aurata). This work demonstrates a fast and efficient strategy for Quantitative Trait Loci (QTL) detection in the gilthead sea bream, a non-model but target hermaphrodite fish species. Therefore a comparative mapping approach was performed to query syntenies against two other Perciformes, the European sea bass (Dicentrarchus labrax), a gonochoristic species and the Asian sea bass (Lates calcarifer) a protandrous hermaphrodite. In this manner two significant QTLs, one QTL affecting both body weight and sex and one QTL affecting sex, were detected on the same linkage group. The co-segregation of the two QTLs provides a genomic base to the observed genetic correlation between these two traits in sea bream as well as in other teleosts. The identification of QTLs linked to sex reversal and growth, will contribute significantly to a better understanding of the complex nature of sex determination in S. aurata where most individuals reverse to the female sex at the age of two years through development and maturation of the ovarian portion of the gonad and regression of the testicular area. [Genomic sequences reported in this manuscript have been submitted to GenBank under accession numbers HQ021443–HQ021749.]

Genetic differentiation and phylogenetic relationships among Greek Salmo trutta L. (brown trout) populations as revealed by RFLP analysis of PCR amplified mitochondrial DNA segments.

The genetic differentiation and phylogenetic relationships of 13 populations (11 from Greece, one from Albania and one from France) of brown trout (Salmo trutta L.) were investigated at the mtDNA level. RFLP analysis of four segments of mitochondrial DNA (D-loop, cytochrome b and ND-5/6) amplified by PCR was used. Seven of 14 restriction endonucleases were found to detect variability in the ND-5/6 regions, whereas four and two out of 17 and 16 were polymorphic in the D-loop and cytochrome b, respectively. Eleven different haplotypes were observed. The observed interpopulation diversity was very high (mean = 1.65 per cent), whereas the intrapopulation diversity was low in most cases (mean = 0.063 per cent). Five phylogenetic assemblages were identified. The results demonstrate that Greece is one of the regions where brown trout display very high levels of genetic diversity. Most populations were genetically very distinct, possessing private mtDNA genotypes. Therefore, they represent unique gene pools which may warrant individual recognition for conservation and management. The genetic relationships among populations suggested by mtDNA data were not in accordance with allozyme data. This study illustrates the importance of considering the historical biogeography of a species in order to understand its population genetic structure. It also reinforces the view that mtDNA analysis represents a powerful tool to study past and present demographic phenomena.

Genetic Identification and Phylogeny of Three Species of the Genus Trachurus Based on Mitochondrial DNA Analysis

The genetic identification and the phylogenetic relationships of 3 European species of the genus Trachurus (T. trachurus, T. mediterraneus, and T. picturatus) across their geographical distribution, have been investigated by mitochondrial DNA analysis. Both cytochrome b and 16S ribosomal DNA sequence analysis revealed the existence of several species-specific positions that distinguish the 3 studied species. Genetic distances between the species indicated that T. mediterraneus and T. picturatus are more closely related than T. trachurus. Similar topologies have been produced by neighbor-joining, maximum-likelihood, and maximum-parsimony trees, and they were in accordance with previous taxonomic classification. Internucleotide and intranucleotide diversity of T. picturatus was 2 times higher than that of T. mediterraneus and T. trachurus, possibly owing to the low levels of fishing pressure for T. picturatus. This is the first report of the phylogenetic relationships of the 3 Trachurus species and provides a possible scenario of the time of divergence related to the closure of the Gibraltar Straits. In addition, the present results can be used for genetic identification of the 3 species, even from the early stage of eggs, and for detection of commercial fraud.

Genetic divergence and phylogenetic relationships among Salmo trutta L. (brown trout) populations from Greece and other European countries

In order to clarify the genetic structure and the phylogenetic relationships among brown trout (Salmo trutta) populations from Greece and other European countries starch gel electrophoresis was used. The populations come from various rivers from Greece and other European countries, flowing to the Atlantic or to the Mediterranean. Eleven enzymic systems were investigated. These correspond to 26 putative loci. A high degree of genetic polymorphism was found. The percentage of polymorphic loci ranged from 3.8 to 34.6 and the degree of expected heterozygosity from 0.016 to 0.1. F-statistics and clustering analyses indicated the existence of a high degree of differentiation. This differentiation is mainly between the Atlantic and the Mediterranean populations. Furthermore the Mediterranean populations seem to be divided into two groups. One includes the western Mediterranean populations and populations from western Greece and the other north-eastern Greek populations. The latter seem to be related to other Balkan populations and probably to Danubian or Black Sea populations. These results support the idea of two different lineages of Mediterranean brown trout populations, one of which is probably of aboriginal origin.

Phylogenetic relationships among four species of Mullidae (Perciformes) inferred from DNA sequences of mitochondrial cytochrome b and 16S rRNA genes

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among four species of mullids. Approximately 238bp of the mitochondrial 16S ribosomal RNA (rRNA) and 261bp of the cytochrome b (cytb) genes were sequenced from representatives of three mullid genera (Mullus, Upeneus, Pseudopeneus), present in the Mediterranean Sea. Trees were constructed using three methods: maximum likelihood (ML), neighbor joining (NJ) and parsimony (MP). The results of the analyses of these data together with published data of the same mtDNA segments of two other perciform species (Sparus aurata, Perca fluviatilis), support the previous taxonomic classification of the three genera examined, as well as the classification of the two red mullet species in the same genus.

Mitochondrial DNA Sequence Variation in Greeks

Mitochondrial DNA (mtDNA) control region sequences were determined in 54 unrelated Greeks, coming from different regions in Greece, for both segments HVR-I and HVR-II. Fifty-two different mtDNA haplotypes were revealed, one of which was shared by three individuals. A very low heterogeneity was found among Greek regions. No one cluster of lineages was specific to individuals coming from a certain region. The average pairwise difference distribution showed a value of 7.599. The data were compared with that for other European or neighbor populations (British, French, Germans, Tuscans, Bulgarians, and Turks). The genetic trees that were constructed revealed homogeneity between Europeans. Median networks revealed that most of the Greek mtDNA haplotypes are clustered to the five known haplogroups and that a number of haplotypes are shared among Greeks and other European and Near Eastern populations.

Genetic differentiation and phylogenetic relationships among Greek Chub Leuciscus cephalus L. (Pisces, Cyprinidae) populations as revealed by RFLP analysis of mitochondrial DNA

Abstract The phylogenetic and systematic relationships among 12 populations of Greek chub ( Leuciscus cephalus L.) were investigated using RFLP analysis of two PCR-amplified mtDNA segments (D-loop and cytochrome b). Three out of eight, and nine out of ten restriction enzymes were polymorphic for D-loop and cytochrome b, respectively. Twenty-one different haplotypes were detected. Estimated nucleotide sequence divergence among the 21 genotypes ranged from 0.313 to 6.79%; the mean value of sequence divergence among populations was 3.421±0.00%. The calculated N ST value of 0.94, indicates high interpopulation diversity. Dendrograms based on haplotypes as well as those of populations, support the previous recognition of different subspecies, but these mtDNA data are not concordant with previous allozyme data among these fish. Our results suggest that to clarify phylogenetic relationships multiple genetic systems need to be examined.

Quantitative trait loci for body growth and sex determination in the hermaphrodite teleost fish Sparus aurata L.

Gilthead sea bream (Sparus aurata L.) is an important marine fish in Mediterranean aquaculture. Sex determination by age and/or body weight is a critical life-history trait, the genetic basis for which is largely unknown in this sequential hermaphrodite species. Herein, we performed a partial genome scan to map quantitative trait loci (QTL) affecting body weight and sex using 74 informative microsatellite markers from 10 paternal half-sib families to construct nine linkage groups (LG). In total, four growth-related QTL (two chromosome-wide and two genome-wide) and six QTL related to sex determination (three pairs in three different LGs) were detected (two chromosome-wide and one genome-wide). The proportion of phenotypic variation explained by the body-weight QTL ranged from 9.3% to 17.2%, showing their potential for use in marker-assisted selection. The results obtained offer solid ground to investigate the structure and function of the genomic regions involved in the mechanisms of sex reversal.

Genetic characterization of brown trout (Salmo trutta) populations from the Southern Balkans using mtDNA sequencing and RFLP analysis

In the present study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to survey variation in a 1450-bp mitochondrial DNA (mtDNA) segment which comprises part of the cytochrome oxidase III (COIII) and ATPase subunit VI genes in 8 brown trout (Salmo trutta L) populations from the southern Balkans. In addition, a 300xa0bp fragment at the 5′ end of the control region was sequenced from representatives of the populations studied providing the opportunity to assign PCR-RFLP haplotypes into major phylogenetic lineages (i.e. Atlantic, Danubian, marmoratus, Adriatic and Mediterranean). The level of polymorphism found in the 1450xa0bp segment suggests that this PCR-RFLP assay may be useful for future diagnostic analyses of mitochondrial DNA in brown trout populations. A reduced within-population genetic variability but considerable among-population differentiation was observed. The results are in accordance with previous data on phylogeography of Mediterranean brown trout suggesting that mitochondrial DNA haplotypes are distributed in a mosaic pattern as a consequence of a complex evolutionary history. The present study shows that brown trout populations from the Southern Balkans are highly divergent and possess a unique genetic profile that should be taken into account when establishing conservation management programs.